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1.
Nat Commun ; 12(1): 3908, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162850

RESUMO

Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, "AsCas12a Ultra", that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.


Assuntos
Acidaminococcus/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Endonucleases/metabolismo , Edição de Genes/métodos , Acidaminococcus/genética , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Células Cultivadas , Endonucleases/genética , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Jurkat , Células Matadoras Naturais/metabolismo , Reprodutibilidade dos Testes , Linfócitos T/metabolismo
2.
J Biol Chem ; 296: 100294, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33755021

RESUMO

Electron bifurcation exploits high energetic states to drive unfavorable single electron reactions and determining the overall mechanism governing these electron transfers represents an arduous task. Using extensive stopped-flow spectroscopy and kinetic simulations, Sucharitakul et al. now explore the bifurcation mechanism of the electron transfer flavoprotein EtfAB from the anaerobic gut bacterium Acidaminococcus fermentans. Strikingly, they illustrated that catalysis is orchestrated by a negatively charged radical, α-FAD, that inhibits further reductions and features an atypical inverted kinetic isotope effect. These results provide additional insight behind electron transfers that are prevalent within multienzyme governed reactions.


Assuntos
Transporte de Elétrons , Acidaminococcus/metabolismo , Proteínas de Bactérias/metabolismo , Catálise , Elétrons , Metabolismo Energético , Flavina-Adenina Dinucleotídeo/metabolismo , Cinética , Oxirredução
3.
J Biol Chem ; 296: 100124, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33239361

RESUMO

Electron bifurcation uses free energy from exergonic redox reactions to power endergonic reactions. ß-FAD of the electron transfer flavoprotein (EtfAB) from the anaerobic bacterium Acidaminococcus fermentans bifurcates the electrons of NADH, sending one to the low-potential ferredoxin and the other to the high-potential α-FAD semiquinone (α-FAD•-). The resultant α-FAD hydroquinone (α-FADH-) transfers one electron further to butyryl-CoA dehydrogenase (Bcd); two such transfers enable Bcd to reduce crotonyl-CoA to butyryl-CoA. To get insight into the mechanism of these intricate reactions, we constructed an artificial reaction only with EtfAB containing α-FAD or α-FAD•- to monitor formation of α-FAD•- or α-FADH-, respectively, using stopped flow kinetic measurements. In the presence of α-FAD, we observed that NADH transferred a hydride to ß-FAD at a rate of 920 s-1, yielding the charge-transfer complex NAD+:ß-FADH- with an absorbance maximum at 650 nm. ß-FADH- bifurcated one electron to α-FAD and the other electron to α-FAD of a second EtfAB molecule, forming two stable α-FAD•-. With α-FAD•-, the reduction of ß-FAD with NADH was 1500 times slower. Reduction of ß-FAD in the presence of α-FAD displayed a normal kinetic isotope effect (KIE) of 2.1, whereas the KIE was inverted in the presence of α-FAD•-. These data indicate that a nearby radical (14 Å apart) slows the rate of a hydride transfer and inverts the KIE. This unanticipated flavin chemistry is not restricted to Etf-Bcd but certainly occurs in other bifurcating Etfs found in anaerobic bacteria and archaea.


Assuntos
Acidaminococcus/metabolismo , Proteínas de Bactérias/metabolismo , Flavoproteínas Transferidoras de Elétrons/metabolismo , Flavinas/metabolismo , Transporte de Elétrons , Cinética , Oxirredução , Filogenia
4.
FEBS J ; 288(3): 1008-1026, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32329961

RESUMO

The flavin-based electron bifurcation (FBEB) system from Acidaminococcus fermentans is composed of the electron transfer flavoprotein (EtfAB) and butyryl-CoA dehydrogenase (Bcd). α-FAD binds to domain II of the A-subunit of EtfAB, ß-FAD to the B-subunit of EtfAB and δ-FAD to Bcd. NADH reduces ß-FAD to ß-FADH- , which bifurcates one electron to the high potential α-FAD•- semiquinone followed by the other to the low potential ferredoxin (Fd). As deduced from crystal structures, upon interaction of EtfAB with Bcd, the formed α-FADH- approaches δ-FAD by rotation of domain II, yielding δ-FAD•- . Repetition of this process leads to a second reduced ferredoxin (Fd- ) and δ-FADH- , which reduces crotonyl-CoA to butyryl-CoA. In this study, we measured the redox properties of the components EtfAB, EtfaB (Etf without α-FAD), Bcd, and Fd, as well as of the complexes EtfaB:Bcd, EtfAB:Bcd, EtfaB:Fd, and EftAB:Fd. In agreement with the structural studies, we have shown for the first time that the interaction of EtfAB with Bcd drastically decreases the midpoint reduction potential of α-FAD to be within the same range of that of ß-FAD and to destabilize the semiquinone of α-FAD. This finding clearly explains that these interactions facilitate the passing of electrons from ß-FADH- via α-FAD•- to the final electron acceptor δ-FAD•- on Bcd. The interactions modulate the semiquinone stability of δ-FAD in an opposite way by having a greater semiquinone stability than in free Bcd.


Assuntos
Acidaminococcus/metabolismo , Proteínas de Bactérias/metabolismo , Benzoquinonas/metabolismo , Butiril-CoA Desidrogenase/metabolismo , Flavoproteínas Transferidoras de Elétrons/metabolismo , Flavinas/metabolismo , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Proteínas de Bactérias/química , Benzoquinonas/química , Butiril-CoA Desidrogenase/química , Transporte de Elétrons , Flavoproteínas Transferidoras de Elétrons/química , Elétrons , Ferredoxinas/química , Ferredoxinas/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Modelos Biológicos , Oxirredução , Ligação Proteica , Espectrofotometria
5.
Nat Biotechnol ; 39(1): 94-104, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32661438

RESUMO

Cas12a RNA-guided endonucleases are promising tools for multiplexed genetic perturbations because they can process multiple guide RNAs expressed as a single transcript, and subsequently cleave target DNA. However, their widespread adoption has lagged behind Cas9-based strategies due to low activity and the lack of a well-validated pooled screening toolkit. In the present study, we describe the optimization of enhanced Cas12a from Acidaminococcus (enAsCas12a) for pooled, combinatorial genetic screens in human cells. By assaying the activity of thousands of guides, we refine on-target design rules and develop a comprehensive set of off-target rules to predict and exclude promiscuous guides. We also identify 38 direct repeat variants that can substitute for the wild-type sequence. We validate our optimized AsCas12a toolkit by screening for synthetic lethalities in OVCAR8 and A375 cancer cells, discovering an interaction between MARCH5 and WSB2. Finally, we show that enAsCas12a delivers similar performance to Cas9 in genome-wide dropout screens but at greatly reduced library size, which will facilitate screens in challenging models.


Assuntos
Proteínas de Bactérias , Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas/genética , Endodesoxirribonucleases , Edição de Genes/métodos , RNA Guia , Acidaminococcus/genética , Apoptose/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Linhagem Celular Tumoral , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Biblioteca Gênica , Células HEK293 , Humanos , RNA Guia/genética , RNA Guia/metabolismo
6.
Nucleic Acids Res ; 48(9): 5037-5053, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32315032

RESUMO

CRISPR RNA-guided endonucleases (RGEs) cut or direct activities to specific genomic loci, yet each has off-target activities that are often unpredictable. We developed a pair of simple in vitro assays to systematically measure the DNA-binding specificity (Spec-seq), catalytic activity specificity (SEAM-seq) and cleavage efficiency of RGEs. By separately quantifying binding and cleavage specificity, Spec/SEAM-seq provides detailed mechanistic insight into off-target activity. Feature-based models generated from Spec/SEAM-seq data for SpCas9 were consistent with previous reports of its in vitro and in vivo specificity, validating the approach. Spec/SEAM-seq is also useful for profiling less-well characterized RGEs. Application to an engineered SpCas9, HiFi-SpCas9, indicated that its enhanced target discrimination can be attributed to cleavage rather than binding specificity. The ortholog ScCas9, on the other hand, derives specificity from binding to an extended PAM. The decreased off-target activity of AsCas12a (Cpf1) appears to be primarily driven by DNA-binding specificity. Finally, we performed the first characterization of CasX specificity, revealing an all-or-nothing mechanism where mismatches can be bound, but not cleaved. Together, these applications establish Spec/SEAM-seq as an accessible method to rapidly and reliably evaluate the specificity of RGEs, Cas::gRNA pairs, and gain insight into the mechanism and thermodynamics of target discrimination.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/metabolismo , Acidaminococcus/enzimologia , Pareamento Incorreto de Bases , Pareamento de Bases , Proteínas Associadas a CRISPR/genética , DNA/química , DNA/metabolismo , Clivagem do DNA , Deltaproteobacteria/enzimologia , Endodesoxirribonucleases/genética , Mutação , Proteína Homeobox Nanog/genética , Ligação Proteica , RNA/química , Técnica de Seleção de Aptâmeros , Análise de Sequência de DNA , Especificidade por Substrato
7.
J Biol Chem ; 295(17): 5538-5553, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32161115

RESUMO

Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.


Assuntos
Acidaminococcus/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA/genética , Endodesoxirribonucleases/metabolismo , Francisella/enzimologia , Acidaminococcus/genética , Acidaminococcus/metabolismo , Proteínas de Bactérias/genética , Pareamento Incorreto de Bases , Sequência de Bases , Proteínas Associadas a CRISPR/genética , DNA/metabolismo , Clivagem do DNA , Endodesoxirribonucleases/genética , Francisella/genética , Francisella/metabolismo , Edição de Genes/métodos , Expressão Gênica
8.
Bioconjug Chem ; 31(3): 542-546, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32119776

RESUMO

CRISPR-Cas12a, a type-V CRISPR-Cas endonuclease, is an effective genome editing platform. To improve the gene editing efficiency of Cas12a, we rationally designed small molecule enhancers through a combined computational approach. First, we used extensive molecular dynamics (MD) simulations to explore the conformational landscape of Cas12a from Acidaminococcus (AsCas12a), revealing distinct conformational states that could be targeted by small molecules to modulate its genome editing function. We then identified 57 compounds that showed different binding behavior and stabilizing effects on these distinct conformational states using molecular docking. After experimental testing 6 of these 57 compounds, compound 1, quinazoline-2,4(1H,3H)-dione, was found particularly promising in enhancing the AsCas12a-mediated genome editing efficiency in human cells. Compound 1 was shown to act like a molecular "glue" at the interface between AsCas12a and crRNA near the 5'-handle region, thus specifically stabilizing the enzyme-crRNA complex. These results provide a new paradigm for future design of small molecules to modulate the genome editing of the CRISPR-Cas systems.


Assuntos
Sistemas CRISPR-Cas/genética , Desenho de Fármacos , Endodesoxirribonucleases/metabolismo , Edição de Genes/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Acidaminococcus/enzimologia , Endodesoxirribonucleases/química , Simulação de Dinâmica Molecular , Conformação Proteica
9.
J Crohns Colitis ; 14(3): 369-380, 2020 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-31501882

RESUMO

BACKGROUND AND AIMS: A personalized approach to therapy hold great promise to improve disease outcomes. To this end, the identification of different subsets of patients according to the prevalent pathogenic process might guide the choice of therapeutic strategy. We hypothesize that ulcerative colitis [UC] patients might be stratified according to distinctive cytokine profiles and/or to a specific mucosa-associated microbiota. METHODS: In a cohort of clinically and endoscopic active UC patients and controls, we used quantitative PCR to analyse the mucosal cytokine mRNA content and 16S rRNA gene sequencing to assess the mucosa-associated microbiota composition. RESULTS: We demonstrate, by means of data-driven approach, the existence of a specific UC patient subgroup characterized by elevated IL-13 mRNA tissue content separate from patients with low IL-13 mRNA tissue content. The two subsets differ in clinical-pathological characteristics. High IL-13 mRNA patients are younger at diagnosis and have a higher prevalence of extensive colitis than low IL-13 mRNA patients. They also show more frequent use of steroid/immunosuppressant/anti-tumour necrosis factor α therapy during 1 year of follow-up. The two subgroups show differential enrichment of mucosa-associated microbiota genera with a prevalence of Prevotella in patients with high IL-13 mRNA tissue content and Sutterella and Acidaminococcus in patients with low IL-13 mRNA tissue content. CONCLUSION: Assessment of mucosal IL-13 mRNA might help in the identification of a patient subgroup that might benefit from a therapeutic approach modulating IL-13. PODCAST: This article has an associated podcast which can be accessed at https://academic.oup.com/ecco-jcc/pages/podcast.


Assuntos
Colite Ulcerativa , Colo , Interleucina-13/genética , Mucosa Intestinal , RNA Ribossômico 16S/genética , Acidaminococcus/isolamento & purificação , Colite Ulcerativa/classificação , Colite Ulcerativa/genética , Colite Ulcerativa/imunologia , Colite Ulcerativa/terapia , Colo/microbiologia , Colo/patologia , Correlação de Dados , Feminino , Humanos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Conduta do Tratamento Medicamentoso/estatística & dados numéricos , Pessoa de Meia-Idade , Seleção de Pacientes , Prevotella/isolamento & purificação , RNA Mensageiro/genética , Índice de Gravidade de Doença
10.
PLoS Biol ; 17(10): e3000268, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31622337

RESUMO

Imaging dense and diverse microbial communities has broad applications in basic microbiology and medicine, but remains a grand challenge due to the fact that many species adopt similar morphologies. While prior studies have relied on techniques involving spectral labeling, we have developed an expansion microscopy method (µExM) in which bacterial cells are physically expanded prior to imaging. We find that expansion patterns depend on the structural and mechanical properties of the cell wall, which vary across species and conditions. We use this phenomenon as a quantitative and sensitive phenotypic imaging contrast orthogonal to spectral separation to resolve bacterial cells of different species or in distinct physiological states. Focusing on host-microbe interactions that are difficult to quantify through fluorescence alone, we demonstrate the ability of µExM to distinguish species through an in vitro defined community of human gut commensals and in vivo imaging of a model gut microbiota, and to sensitively detect cell-envelope damage caused by antibiotics or previously unrecognized cell-to-cell phenotypic heterogeneity among pathogenic bacteria as they infect macrophages.


Assuntos
Acetobacter/ultraestrutura , Escherichia coli/ultraestrutura , Lactobacillus plantarum/ultraestrutura , Microscopia/métodos , Muramidase/farmacologia , Acetobacter/efeitos dos fármacos , Acidaminococcus/efeitos dos fármacos , Acidaminococcus/ultraestrutura , Animais , Antibacterianos/farmacologia , Parede Celular/química , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Drosophila melanogaster/microbiologia , Escherichia coli/efeitos dos fármacos , Microbioma Gastrointestinal/fisiologia , Humanos , Hidrólise , Lactobacillus plantarum/efeitos dos fármacos , Camundongos , Microscopia/instrumentação , Muramidase/química , Platelmintos/microbiologia , Células RAW 264.7 , Estresse Mecânico , Simbiose/fisiologia , Vancomicina/farmacologia
11.
Angew Chem Int Ed Engl ; 58(48): 17399-17405, 2019 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-31568601

RESUMO

An accurate, rapid, and cost-effective biosensor for the quantification of disease biomarkers is vital for the development of early-diagnostic point-of-care systems. The recent discovery of the trans-cleavage property of CRISPR type V effectors makes CRISPR a potential high-accuracy bio-recognition tool. Herein, a CRISPR-Cas12a (cpf1) based electrochemical biosensor (E-CRISPR) is reported, which is more cost-effective and portable than optical-transduction-based biosensors. Through optimizing the in vitro trans-cleavage activity of Cas12a, E-CRIPSR was used to detect viral nucleic acids, including human papillomavirus 16 (HPV-16) and parvovirus B19 (PB-19), with a picomolar sensitivity. An aptamer-based E-CRISPR cascade was further designed for the detection of transforming growth factor ß1 (TGF-ß1) protein in clinical samples. As demonstrated, E-CRISPR could enable the development of portable, accurate, and cost-effective point-of-care diagnostic systems.


Assuntos
Aptâmeros de Nucleotídeos/química , Sistemas CRISPR-Cas/genética , DNA Viral/química , Papillomavirus Humano 16/genética , Ácidos Nucleicos Imobilizados/química , Parvovirus/genética , Acidaminococcus/genética , Técnicas Biossensoriais , Clivagem do DNA , Técnicas Eletroquímicas , Eletrodos , Humanos , Limite de Detecção , Células-Tronco Mesenquimais , Sensibilidade e Especificidade , Propriedades de Superfície , Fator de Crescimento Transformador beta1/análise , Fator de Crescimento Transformador beta1/metabolismo
12.
Nat Commun ; 10(1): 3556, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391465

RESUMO

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. The 3272-26A>G and 3849+10kbC>T CFTR mutations alter the correct splicing of the CFTR gene, generating new acceptor and donor splice sites respectively. Here we develop a genome editing approach to permanently correct these genetic defects, using a single crRNA and the Acidaminococcus sp. BV3L6, AsCas12a. This genetic repair strategy is highly precise, showing very strong discrimination between the wild-type and mutant sequence and a complete absence of detectable off-targets. The efficacy of this gene correction strategy is verified in intestinal organoids and airway epithelial cells derived from CF patients carrying the 3272-26A>G or 3849+10kbC>T mutations, showing efficient repair and complete functional recovery of the CFTR channel. These results demonstrate that allele-specific genome editing with AsCas12a can correct aberrant CFTR splicing mutations, paving the way for a permanent splicing correction in genetic diseases.


Assuntos
Acidaminococcus/genética , Proteínas Associadas a CRISPR/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/terapia , Edição de Genes/métodos , Alelos , Proteínas de Bactérias/genética , Biópsia , Técnicas de Cultura de Células , Linhagem Celular , Fibrose Cística/genética , Fibrose Cística/patologia , Endonucleases/genética , Humanos , Intestinos/patologia , Organoides , Mutação Puntual , Sítios de Splice de RNA/genética , Splicing de RNA/genética
13.
Nat Methods ; 16(9): 887-893, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31406383

RESUMO

The ability to modify multiple genetic elements simultaneously would help to elucidate and control the gene interactions and networks underlying complex cellular functions. However, current genome engineering technologies are limited in both the number and the type of perturbations that can be performed simultaneously. Here, we demonstrate that both Cas12a and a clustered regularly interspaced short palindromic repeat (CRISPR) array can be encoded in a single transcript by adding a stabilizer tertiary RNA structure. By leveraging this system, we illustrate constitutive, conditional, inducible, orthogonal and multiplexed genome engineering of endogenous targets using up to 25 individual CRISPR RNAs delivered on a single plasmid. Our method provides a powerful platform to investigate and orchestrate the sophisticated genetic programs underlying complex cell behaviors.


Assuntos
Sistemas CRISPR-Cas , Endonucleases/metabolismo , Edição de Genes , Redes Reguladoras de Genes , Engenharia Genética , Genoma Humano , RNA Guia/genética , Acidaminococcus/enzimologia , Endonucleases/genética , Células HEK293 , Humanos , Plasmídeos/genética , Ativação Transcricional
14.
Elife ; 82019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31397669

RESUMO

CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the L. bacterium Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The Ac. species Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.


Assuntos
Acidaminococcus/enzimologia , Bacteriófagos/crescimento & desenvolvimento , Sistemas CRISPR-Cas/efeitos dos fármacos , Clostridiales/enzimologia , Inibidores Enzimáticos/metabolismo , Interações Hospedeiro-Parasita , Proteínas Virais/metabolismo , Acidaminococcus/virologia , Clostridiales/virologia , Evolução Molecular
16.
Food Res Int ; 121: 568-576, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31108782

RESUMO

There is little information on the microbial communities associated with modified atmosphere (MA)-packaged live mussels. There is also a dearth of information on how pre-packaging depuration modifies the microbial communities and spoilage of live mussels. Amplicon sequencing was used to describe spoilage microbial succession in MA-packaged live mussels during storage at 4 °C. Proteobacteria, Cyanobacteria and Firmicutes were the three major phyla observed in the mussel meat and pouch water of undepurated and depurated mussels. Among these phyla, Cyanobacteria was more predominant on day 0 in mussel meat of undepurated and depurated mussels while Proteobacteria was predominant in commercially-depurated mussels. Synechococcus was apparently dominant on days 0-7 in the meat of undepurated mussels and days 0-10 in depurated mussels. Shewanella was dominant on day 0 in commercially-depurated mussels and dominant on day 15 in undepurated while Acidaminococcus was dominant in depurated mussels on day 15. Psychromonas was observed to be dominant in commercially-depurated mussels on day 7 and further shifted to Acinetobacter by day 10 and 15. In the pouch water, Acinetobacter was dominant throughout the storage days in undepurated mussels while Psychrobacter was predominant in both depurated and commercially-depurated mussels. This study demonstrated the impact of depuration on the microbiota and the spoilage mechanism of MA-packaged live mussels. Shewanella was easily removed through depuration. However, spoilage bacteria such as Acidaminococcus could not be easily removed although they are not important at the beginning but grew at the end. Pouch water contributed suitable biological medium for the growth of Acinetobacter and Psychrobacter and both enhanced the growth of spoilage bacteria such as Shewanella and Acidaminococcus.


Assuntos
Bivalves/microbiologia , Microbiologia de Alimentos , Embalagem de Alimentos , Microbiota/genética , RNA Ribossômico 16S/genética , Alimentos Marinhos/microbiologia , Acidaminococcus , Animais , Atmosfera , Austrália , Bactérias/classificação , Bactérias/genética , Biodiversidade , Cianobactérias , Carne/microbiologia , Shewanella
17.
Nat Commun ; 10(1): 2092, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064995

RESUMO

The CRISPR effector protein Cas12a has been used for a wide variety of applications such as in vivo gene editing and regulation or in vitro DNA sensing. Here, we add programmability to Cas12a-based DNA processing by combining it with strand displacement-based reaction circuits. We first establish a viable strategy for augmenting Cas12a guide RNAs (gRNAs) at their 5' end and then use such 5' extensions to construct strand displacement gRNAs (SD gRNAs) that can be activated by single-stranded RNA trigger molecules. These SD gRNAs are further engineered to exhibit a digital and orthogonal response to different trigger RNA inputs-including full length mRNAs-and to function as multi-input logic gates. We also demonstrate that SD gRNAs can be designed to work inside bacterial cells. Using such in vivo SD gRNAs and a DNase inactive version of Cas12a (dCas12a), we demonstrate logic gated transcriptional control of gene expression in E. coli.


Assuntos
Proteínas Associadas a CRISPR/genética , Edição de Genes/métodos , RNA Guia/genética , Acidaminococcus/genética , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Plasmídeos/genética , Transformação Bacteriana
18.
FEMS Microbiol Lett ; 366(8)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31004485

RESUMO

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nuclease Acidaminococcus sp. Cas12a (AsCas12a, also known as AsCpf1) has become a popular alternative to Cas9 for genome editing and other applications. AsCas12a has been associated with a TTTV protospacer-adjacent motif (PAM) as part of target recognition. Using a cell-free transcription-translation (TXTL)-based PAM screen, we discovered that AsCas12a can also recognize GTTV and, to a lesser degree, GCTV motifs. Validation experiments involving DNA cleavage in TXTL, plasmid clearance in Escherichia coli, and indel formation in mammalian cells showed that AsCas12a was able to recognize these motifs, with the GTTV motif resulting in higher cleavage efficiency compared to the GCTV motif. We also observed that the -5 position influenced the activity of DNA cleavage in TXTL and in E. coli, with a C at this position resulting in the lowest activity. Together, these results show that wild-type AsCas12a can recognize non-canonical GTTV and GCTV motifs and exemplify why the range of PAMs recognized by Cas nucleases are poorly captured with a consensus sequence.


Assuntos
Acidaminococcus/genética , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Endonucleases/genética , Motivos de Nucleotídeos , Acidaminococcus/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Domínio Catalítico , Clivagem do DNA , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Escherichia coli/genética , Edição de Genes , Células HEK293 , Humanos , Plasmídeos/genética
19.
Nat Biotechnol ; 37(3): 276-282, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30742127

RESUMO

Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases1 has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)2. To address this limitation, we engineered an enhanced Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants3 to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing.


Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas/genética , Endonucleases/genética , Edição de Genes , Ribonucleoproteínas/genética , Acidaminococcus/enzimologia , Epigênese Genética/genética , Células HEK293 , Humanos , Mutação , Linfócitos T/metabolismo
20.
Mol Cell ; 71(5): 816-824.e3, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30078724

RESUMO

Class 2 CRISPR-Cas nucleases are programmable genome editing tools with promising applications in human health and disease. However, DNA cleavage at off-target sites that resemble the target sequence is a pervasive problem that remains poorly understood mechanistically. Here, we use quantitative kinetics to dissect the reaction steps of DNA targeting by Acidaminococcus sp Cas12a (also known as Cpf1). We show that Cas12a binds DNA tightly in two kinetically separable steps. Protospacer-adjacent motif (PAM) recognition is followed by rate-limiting R-loop propagation, leading to inevitable DNA cleavage of both strands. Despite functionally irreversible binding, Cas12a discriminates strongly against mismatches along most of the DNA target sequence. This result implies substantial reversibility during R-loop formation-a late transition state-and defies common descriptions of a "seed" region. Our results provide a quantitative basis for the DNA cleavage patterns measured in vivo and observations of greater reported target specificity for Cas12a than for the Cas9 nuclease.


Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/genética , RNA Guia/genética , Acidaminococcus/genética , Proteínas de Bactérias/genética , Clivagem do DNA , Edição de Genes/métodos , Humanos , Cinética , Conformação de Ácido Nucleico , Ligação Proteica
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